CCMB: Can alpha chain of clusterin protein control weight, fat gain in animals?

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Dr. Ramakrishnan (left), Dr. Mohan Rao (centre) and Suvarsha Matukumalli studied rats injected with beta chain and found fat accumulation from day two onwards.

The two chains of clusterin protein, which are normally expressed in several tissues and can be found in several body fluids, when present together tend to lower lipid levels but administration of one of its chains — alpha or beta — results in completely different outcomes. Cells treated with a recombinant beta chain tend to accumulate fat while cells treated with an alpha chain showed no increase in lipid accumulation. Rabbits administered with a recombinant beta chain showed nearly 40% increase in weight while animals given an alpha chain showed no such increase. The results were published in the journal Scientific Reports.

“Two chains of clusterin when present together tend to decrease body weight but one of the two chains (beta clusterin) increases body weight. This is quite unusual,” says Dr. Ch. Mohan Rao from the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, and the corresponding author of the paper. “So the alpha chain should ideally be compensating for increase in body weight. But the alpha chain does not do that.”

Rats gained lean mass not fat

“While excess energy gets accumulated in the form of fat when beta chain was injected into rats, we did not see this in the case of alpha chain. One possibility is that the alpha chain helps in the metabolism of food in such a way that fat does not accumulate,” he says. “Dissected rats that were given alpha chain showed increased levels of lean mass.”

Apparently, there was no difference in the food intake between animals treated with alpha or beta chain. “It means that weight increase can happen even when there is no increase in food intake. It is the energy management by the body that is important. And alpha chain seems to modulate metabolism in such a way to promote energy expenditure and thus prevent fat accumulation,” he says.

The effect of alpha and beta chains were tested on myoblast cells, fibroblast and cancer cells. The individual chains were injected into rabbits as well. “In my lab we study the effect of small heat shock protein on health and disease. To raise antibody for clusterin we injected the chains separately into rabbits. One set of rabbits was gaining weight while the other did not. That’s when we investigated the reasons. The animal-house in-charge noticed the change in the animals,” recalls Dr. Rao.

Rats too gained weight

Though the effects of the two chains were seen in rabbits, the researchers turned to rats as more animals were required for investigating the effect of individual chains on animals.

“We could see fat accumulation in cells from day two onwards. We observed for 10 days and fat accumulation continued for all the 10 days; we could study cells continuously only for 10 days,” says Suvarsha Rao Matukumalli from CCMB and the first author of the paper. “In the case of animals injected with beta chain, fat accumulation continued for four-five months. The controls and animals given alpha chain did not show weight or fat gain.”

When cells were administered both the chains simultaneously, the cells did not accumulate fat for two-three days but started thereafter. “Fat accumulation was not as much as when only the beta chain was given but fat accumulation nevertheless continued,” says Ms. Matukumalli. But the effect of both the chains in animals was quite different. “When we introduced both alpha and beta chains together in animals we did not see any weight gain. The animals were very much like the controls,” she says. “Only large-scale, in-depth studies can reveal if alpha chain prevents weight gain.”

Published in The Hindu on March 19, 2017

Ostriches existed in India over 25,000 years ago, DNA evidence shows

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Scientists found 92% genetic similarity between the fossil eggshell samples and Struthio camelus, an ostrich species found in Africa.

Based on a DNA analysis of a fossilised eggshell fragment of ostrich, Indian researchers have for the first time found molecular evidence to confirm the presence of these birds in India more than 25,000 years ago. Scientists found 92% genetic similarity between the fossil eggshell samples and Struthio camelus, an ostrich species found in Africa. The results were published in the journal PLOS ONE.

Eggshell fragments of ostrich discovered from India before have been studied using morphological features, which is insufficient to confirm the existence of ostrich in India. This is the first time that molecular evidence indicating their presence in India has been obtained.

Proves the continental drift

The ostrich sample analysed has close proximity to the African ostrich species and underlines the movement of these birds between Africa and India before the Indian landmass drifted away from Africa.

Eleven fossilised eggshell samples from eight archaeological sites in Rajasthan, Gujarat and Madhya Pradesh were studied. The DNA was isolated from five samples and a tiny portion (43 base pairs) of a highly-conserved mitochondrial region was amplified and then sequenced from one of the samples. The other four samples had less than 30 base pairs and hence were not included in the study.

“We could amplify only a small portion of the DNA as the sample was highly degraded. It is scientifically challenging to isolate DNA from ancient samples. And in this case, the samples were exposed to hostile environment,” says Dr. Kumarasamy Thangaraj from the Centre for Cellular and Molecular Biology (CCMB), Hyderabad and one of the authors of the paper.

This is a hard shell to crack

“It is very difficult to study ancient DNA as it is often broken into small fragments. In this case, the DNA was highly fragmented. So we could amplify only a smaller overlapping DNA fragment,” Dr. Thangaraj says. “We are happy that we could get this much information considering that the sample was very old and not well-preserved.”

It is the first time that long-term DNA preservation in fossil eggshell collected from tropical environments, as seen in India, has been studied.

“Eggshells are a better substrate than bones for preserving ancient DNA. The intracrystalline structure of the shell minimises microbial contamination,” says Sonal Jain from the Department of Biotechnology, Indian Institute of Technology (IIT) Roorkee and the first author of the paper.

“X-ray diffraction and electron backscattering diffraction studies helped in finding which eggshells were good and which were bad. The shells are made of calcium carbonate. If calcium changes to magnesium and if there is more magnesium then the sample is more degraded,” says Jain.

Published in The Hindu on March 9, 2017

CCMB researchers unravel the skin colour genetics of Indians

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(From left) Dr. Kumarasamy Thangaraj, Dr. S. Nizamuddin, Biswajit Roy and Dr. Niraj Rai have found a new SNP that is associated with skin colour in Indians.

A study of skin colour of 1,167 people belonging to 27 ethnic groups living in Uttar Pradesh and Bihar found that social structure defined by the caste system has a “profound influence on skin pigmentation”. The skin colour was found to vary significantly among ethnic groups and social categories studied.

Accordingly, Brahmins of Uttar Pradesh have the fairest skin while Manjhis (Majhwars) have the darkest skin (highest skin pigmentation). Bhagats exhibit maximum variation in skin pigmentation. Four social groups — general, scheduled caste, other backward caste and religious group — were studied. The results were published in The Journal of Investigative Dermatology.

The association of rs1426654, a key single nucleotide polymorphism (SNP) in SLC24A5 gene, with skin colour has been well established. In fact, this SNP explains 25-38 per cent of pigmentation differences between Europeans and west Africans. “In addition to rs1426654, our study found another SNP (rs2470102) to be significantly associated with skin colour in the Indian population,” says Dr. Kumarasamy Thangaraj from the Centre for Cellular and Molecular Biology (CCMB), Hyderabad and the corresponding author of the paper.

The new SNP was found to independently affect skin pigmentation variation among the Indian population. While the well known SNP (rs1426654) has been found to have a significantly larger effect on skin colour ranging from Europeans

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Brahmins of Uttar Pradesh have the fairest skin and Manjhis have the darkest skin.

to western Africans, the new SNP that the Indian researchers discovered is predominant in India/Asia. But both SNPs taken together are able to better explain the variation in skin colour among the Indian population than each of the SNP individually. The two SNPs together account for over 38 per cent of the variability in skin colour in the Indian population.

The researchers compared the skin colour with the genotype of the individuals. Homozygous (similar) mutant alleles tend to cause lighter skin colour while homozygous wild alleles tend to cause darker skin colour. “So those with homozygous mutant alleles of the new SNP had fairer skin compared with those who had homozygous wild type alleles,” he says. The difference in skin colour persisted even when the contribution by the well known SNP was adjusted. “This shows that the new SNP has an independent effect on skin colour,” says Dr. Thangaraj.

People who had a combination of similar (homozygous) mutant alleles of both the new and the known SNP had the fairest skin; they are said to belong to H1 haplotype. The frequency of the H1 haplotype was far higher (96 per cent) in people with lighter skin than in darker skin (37 per cent). “A particular haplotype is not exclusive to a social category. Though the frequency is less, we do find H1 haplotype in dark skinned social category. This is why we have fair skinned people even in the dark skin social category and dark skinned people in the otherwise fair skin social category,” he says.

India-wide study

In a subsequent study, Dr. Thangaraj and his team genotyped 1,825 individuals belonging to 52 diverse populations in India. They found the allele frequencies of the two SNPs were similar among the Indian population and spread across the population. “Like in Uttar Pradesh and Bihar, the proportion of both mutant and wild homozygous alleles is distributed in differently frequencies in different populations across the Indian population. Also, the H1 haplotype was not exclusive to any particular population or social category,” he says.

The study found that ultraviolet radiation-based selection model alone cannot account for the entire range of variation in skin colour seen in the Indian population. Rather, it is interplay between selection pressure for lighter skin in response to relatively less sunlight and admixture of the two founding populations of India.

Published in The Hindu on November 20, 2016

One more reason to avoid consanguineous marriages

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A ring chromosome was seen in a two-year-old child of a first-cousin marriage, says Dr. Lakshmi Kandukuri (right). 

A body of evidence shows that children born out of consanguineous marriages suffer from several congenital disorders. This is particularly so in the case of children born out of union involving first cousins. The problem in close relative marriages surfaces when a person carries a defect in any of the genes associated with some form of disease and marries a person from the same kindred who also shares the defect. The child inherits two copies of this faulty gene, and thus has the disease.

Now, a study published recently in the journal Cytogenetic and Genome Research reports the presence of a ring chromosome in a two-year-old child of a first-cousin marriage.  The child died when he was seven years old.

Ring chromosomes are rare unbalanced chromosomal abnormalities that occur in about 1 in 50,000 foetuses, says the paper. Ring chromosomes occur when the tips of the chromosome are broken at both ends followed by fusion of these ends or telomere-telomere fusion of the chromosome without the loss of genetic material.

“In this case, the telomere was intact but the telomeric repeats may have got lost during the formation of the ring chromosome,” says Dr. Lakshmi R. Kandukuri from the Centre for Cellular and Molecular Biology, Hyderabad. One of the chromosomes of the chromosome 9 pair exhibited the ring structure.

As a result of the ring chromosome 9, the child had developmental delay.  “The two-year milestones were not reached on time but were delayed,” she says. The child also had facial dysmorphic features including low-set ears and upward slanting eyes, microcephaly, suffered from seizures and had mild spasticity.

Karyotyping revealed that different chromosomal abnormalities were present in differing proportions.  The anomalies comprised of a ring chromosome 9, two ring chromosomes, a large ring chromosome 9 with two centromeres and finally absence of the ring chromosome itself.

Detailed study of the ring chromosome 9 revealed gene loss due to deletion of a particular segment of the chromosome. However, the loss was seen in only one arm of the abnormal chromosome. Chromosomal microarray analysis revealed a 15.7 Mb deletion in one arm of the chromosome 9. “This region is crucial as it carries genes associated with seizure disorder, speech impairment and intellectual disability,” says Dr. Kandukuri.

Though the observable characteristics of the child could be attributed to the loss of genes encompassed in this region, in-depth studies including molecular characterization of the genes involved are required to elucidate the contributory mechanism and to thus correlate the different features such as developmental delay, facial characteristics, seizure and microcephaly to the genotype, she stressed.

Published in The Hindu on June 20, 2016

Gene responsible for obesity in Indians identified

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Obesity is a multigenic condition. – Photo: R. Prasad

For the first time, Indian researchers have been able to isolate a gene associated with obesity which is specific to the Indian population. This is important, as identifying the genetic determinants of body mass index (BMI) will go a long way in better understanding the biological basis of overweight and obesity.

The aim of a study undertaken by research team led by Dr. Kumarasamy Thangaraj of the Hyderabad-based Centre for Cellular and Molecular Biology (CCMB) was to find a novel locus in Indian population. To do that, they excluded the genes that have already been associated with obesity in other populations. A total of 204 non-smoking subjects free of chronic diseases and belonging to different BMI categories — underweight, normal and overweight and obese — were chosen for the study. The subjects were 20-30 years old.

Nearly one million SNP (single nucleotide polymorphism) markers distributed throughout the genome were analysed. “We found one SNP marker (which is within a gene) of THSD7A was significantly associated with obesity. This gene has not been associated with obesity in the Indian population,” said Dr. Thangaraj. The aim of the study was to find a novel locus in the Indian population. The results were published recently in the International Journal of Obesity.

To reconfirm its role, a replication study involving 655 people belonging to different BMI categories — underweight, normal, overweight and obese — was undertaken. “We found highly significant association between the marker and obesity in the replication study,” he said.

THSD7A is a neural N-glycoprotein, which promotes angiogenesis. Angiogenesis, in turn, modulates obesity, adipose metabolism and insulin sensitivity. And now, the authors have been able to find a correlation and this information can be used for drug target, early diagnosis of obesity and treatment. Explaining how the gene is linked to obesity, Dr. Thangaraj said: “The gene is present in everyone. But when there is a mutation to the gene, there is a likelihood that the person carrying the mutated gene will end up being obese.”

However, the gene mutation is not found in all obese people. Similarly, the gene mutation was also found in very small number of underweight people. “That is because obesity is a multigenic condition,” he explained. Despite being a multigenic condition, people carrying the mutation can always take measures to keep obesity at bay, he said. There is a possibility that the SNP marker of THSD7A may be associated with obesity in other South Asian population.

The genetic affinity Indians have with other South Asian population has already been well documented in a 2009 study. In the 2009 study, a particular gene mutation in Indians was found to increase the risk of heart failure in people with cardiomyopathy. And this mutation was found to be a risk factor implicated in South Asian people with cardiomyopathy too.

Published in The Hindu on August 24, 2015

Study throws light on genetic diversity of Indian population

Published in The Hindu on September 24, 2009

A paper published online today (September 24) in the journal Nature, shows that all diverse groups seen spread out in India today come from two major ancient populations that are genetically divergent.

The two ancient populations are the Ancestral North Indians (ANI) and the Ancestral South Indians (ASI). The study was based on genetic analysis.

While the Ancestral North Indians (ANI) group is genetically close to Middle Easterners, Central Asians, and Europeans, the Ancestral South Indians (ASI) are not related to any group outside India, notes the paper.

The study was undertaken by the Hyderabad-based Centre for Cellular and Molecular Biology (CCMB) and three other institutions in the U.S.

Samples from 132 people representing 25 groups from 15 States and speaking six language families (including two language families from the Andaman Islands) were studied.

The study also looked for genetic variations based on caste — upper and lower caste — from two the States of Uttar Pradesh and Andhra Pradesh.

The study found the groups (seen today) that emerged from the two ancient populations have distinct genetic affinity. “The populations (groups) that emerged from ANI show 40-80 per cent genetic affinity to European population.

“But the populations that emerged from ASI don’t show any affinity to any population outside India,” said Dr. Kumarasamy Thangaraj, Senior Scientist at CCMB, and one of the authors of the paper. “The Hyshi and Ao Nage population from north-east India show genetic affinity to the Chinese.”

The indigenous population seen in the Andamans have more affinity to the ASI. “Otherwise, they have no relationship with any other population anywhere in the world,” said Dr. Thangaraj.

Unlike the European and Chinese population, the Indian population are more scattered in a genetic sense.

Medical implications

While consanguinity is often implicated for many recessive gene disorders in a population, this study found that the “shared descent from a common ancestral population plays a bigger role. This is called as the ‘founder effect.’

Founder effect is nothing but the fact that many groups seen today have descended from a small group of founding individuals, and these founding individuals in turn have been isolated from other groups, genetically speaking.

It is based on this fact that the authors state that the founder effect plays a bigger role. “We propose that the founder effect is responsible for an even higher burden of recessive diseases in India than consanguinity,” the paper states.

Dr. Thangaraj explained the significance of this. A disease can occur due to the presence of a recessive gene due to mutation. In a small population with high endogamy [where people marry within the population], the mutation persists and spreads to more number of people.

After a point of time, a large number of people have the recessive gene and this increases the chance of a child receiving a recessive gene from both the parents and thus becoming diseased.

Existence of caste

Contrary to popular perception by historians that the caste system seen today is an invention of colonialism, the study found scientific evidence to show that “many current distinctions among groups are ancient.”

“The caste system is not recent,” said Dr. Thangaraj. “The social stratification existed right from early human divergence, some 50,000-60,000 years ago when initial settlement happened in India.”

The paper adds a word of caution: “Models in population genetics should be treated with caution. Although they provide an important framework for testing historical hypotheses, they are oversimplifications.”

GM crops’ biosafety testing procedure questioned

Published in The Hindu on June 25, 2008

The way in which the Genetic Engineering and Approval Committee (GEAC) has gone about approving field trials of genetically modified crops, as also the final approval for commercial cultivation, has been questioned by P.M. Bhargava, the former Director of the Hyderabad-based Centre for Cellular and Molecular Biology.

Dr. Bhargava who was appointed a special invitee to the GEAC in February this year at the instance of the Supreme Court to bring about more transparency has been very critical of the regulatory body’s rationale for accepting the results of biosafety studies generated by the applicants.

The minutes of the GEAC meeting held on May 28 has taken note of his disapproval of the way the committee has been relying on the data generated by the applicant and hence its usefulness. However, it has defended itself by noting that accepting the data provided by applicants, as in the case of pharmaceutical companies, is a common practice.

The minutes note that “… it will be unfair to mistrust the samples and data generated by the applicant, without any basis.”

“The clinical trial data [for a drug] is not generated by one lab. And the trials are almost always multicentric [conducted in many locations]. All the procedures of the trials are so well documented and the preclinical trials [toxicity testing done on animals prior to starting the trials in humans] can be replicated by anyone,” stressed Dr. Bhargava. “All these don’t happen in the case of GM safety testing.”

But the biggest difference is that most of the human clinical trials for testing drugs are double blinded. This makes sure that neither the person conducting the trial nor the volunteers will know if he has been given a drug or a placebo. And the volunteer recruitment is done by the centres conducting the trials.

This does not happen in the case of GM testing. None of the tests done were double blinded. The samples were provided by the companies. “Do the institutes conducting the tests have the facility to check if samples provided by the companies are indeed genetically modified samples and not non-GM ones,” he asked.

Despite the numerous checks and balances to ensure human clinical trials are conducted and reported correctly, the number of instances where the pharmaceutical companies engage in misconduct are aplenty.

Many measures have been taken to make the conduct of clinical trials more transparent, thus giving less scope for any malpractice. The pharmaceutical companies based in the U.S. are now required to make clinical trial results available in the government database. “So what happens when no such monitoring mechanisms exists for testing GM crops,” he asked.

“There is a great need for a central dedicated institute for collecting and conducting all GM related safety tests,” he said. “The institutions where the tests were done have not been set up for looking at the safety of GM crops.” There is a pressing need for such an institute as there are more GM food crops lined up for testing.

If the procedure for approving drugs is strict, the basis for approving GM crops should be stringent as food is consumed by animals and a large number of people when compared with drugs. And unlike drugs, GM crops cannot be recalled from the market once they are produced.

“See what happened with hyacinth plants and parthenium weeds,” he said. “There is a world of difference between drugs and GM crops.”